A biochemical study of the biological clock of the fungus, Neurospora crassa, is proposed. We will employ a particular mutant strain, bd, since it has a clear, well-defined rhythm expressed as a rhythmic pattern of spore formation (conidiation). This rhythm is circadian in that its period is 22 hrs, is temperature-compensated, and its phase can be altered by flashes of light. The key idea in this proposal is that mitochondria play a significant role in the generation of the circadian rhythm. The hypothesis is based on several lines of evidence, most notably the finding that oligomycin-resistant mutations oli(r) in subunit #9 (DCCD binding protein) of the mitochondrial ATPase lead to altered period. This finding will be extended by purifying subunit #9 and analyzing its primary structure in a variety of other clock mutants. A second line of studies will be to purify subunit #6, also in the F0 portion of the ATPase complex, and to assess its pantothenic acid content in the cel+ and cel strains. The cel- strain is severely deficient in the amount of pantothenic acid attached to its fatty acid synthetase, and we are proposed to test the possibility that the reason the "clock" in the cel- strain is sensitive to temperature is due to a deficiency in the amount of pan on subunit #6. Since subunits #6 and #9 are both integral membrane proteins, and are unusually hydrophobic, these studies tie in nicely with a third set of proposed studies, i.e., the effects of exogenous fatty acids on the clock mechanism. This will be investigated employing C14 unsaturated fatty acids and determining their metabolic fate and localization in membrane lipids. The 4th and 5th aspects of this proposal are the screening for clock defects of existing mutants, such as Ts and cold-sensitives, etc., and the selection of new mutants, primarily those involved in mitochondrial or lipid metabolism, for possible clock changes. the studies in this proposal are viewed as studies of a basic cellular regulatory mechanism, widespread in eucaryotic cells, and yet completely undescribed in bio-chemical terms. Some molecular facts about the mechanism which generates circadian rhythms should be helpful in understanding related phenomena, such as the control of cellular energetics and cell division.